Introduction

Acute Myeloid Leukemia

Acute Myeloid Leukemia is characterized by an increase in the number of myeloid cells in the marrow and the premature terminal of the maturation of these myeloid cells. This results primarily in hematopoietic insufficiency.

Azacitidine

Azacitidine is a DNA methyltransferase inhibitor (DNMTi) which has been used to treat AML patients. However, it is unclear which genes are regulated or hypomethyalted by Azacitidine. Understanding which genes are regulated by Azacitidine could provide us with a better understanding of …

Study Design

In this study, OCI-AML3 cells were treated with 5-azacytidine, with a paired control. The experiment was done in triplicates and then total RNA was sequenced.

QC

Principal Component Analysis

In PCA, a clustering method is applied to essentially check if the treatment condition is the main variable responsible for variation in the data and to check if our replicates within each condition is similar. From the figure below we can see that the treatment condition accounts for 97% (PC1) of the variation and there is minimal variation between technical replicates (1%; PC2).

Differentially Expressed Genes (DEGs) Analysis

Volcano Plot of DEGs

A volcano plot is typically used to quickly visualize the relationship between the p-value (or statisitcal significance) and log2 fold change in expression at the gene level. Using abritrary parameters ie. fold-change and p-value cut offs, we can visualize the proportion of genes that are signifcant genes and are up or down regulated.

In the figure below, we can see that following treatment with 5-azacytidine, there was not much significantly DEGs and a large majority of the DEGs seem to be downregulated. The parameters used were: adjusted p-value <= 0.05 and absolute fold-change >=1.5.

Fig.2: Volcano Plot of Differentially Expressed Genes in Treated vs Untreated Samples

Fig.2: Volcano Plot of Differentially Expressed Genes in Treated vs Untreated Samples

Heatmap of all DEGs

Through a heatmap we can visualize the differences in gene expression between samples of different treatment conditions and observe for conistency within the treatment group. In the figure below, a significant number of genes are downregulated upon the 5-azacytidine treatment when compared to the control.

Fig.3: Heatmap of differentially expressed genes

Fig.3: Heatmap of differentially expressed genes

Top 20 Over- and Under-expressed DEGs

We then looked at the top 20 over and under expressed DEGs, visuazlied through a heatmap.

Pathway Analysis

GSEA

GSEA is used to have a more holistic view, looking at changes at entire pathways instead of a single gene.

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